Chromatography Forum

Hello,
I'm using a Shimadzu Prominance LC-20AR HPLC/FLU for ciprofloxacin detection. For stock solution I use Vetranal® ciprofloxacin analytical std. in Methanol (not the HCl form).

My problem is, when I try to measure the calibration points repetitions, the standard deviation of my data is bigger than 15%.
I tried with glass and nylon syringe filter with 2-12-24 ml rinsing volume.

The pH of the stock solution is ~5.6 so 48% of ciprofloxacin is in cationic form and 52% is in Zwitterionic form. My HPLC eluent is pH 2.3 in this pH ciprofloxacin is in cationic form.

(HPLC parameters: A: phosphoric acid pH 2,3, B: ACN; 72:28%; 1ml/min; inj.vol.:20 ul; T:35 °C, detector: ex: 280nm em: 450nm, Teknokroma Brisa C18 column)

Do you have any advice what should I try, to stabilize my measurements, or what could be the problem?

Thank you very much.

Have you tried matching the stock calibration solution matrix to the mobile phase? (Diluting your stock with initial mobile phase)

Thank you for your reply.
No, I shouldn't modify the pH of the solutions, because later I will use it to adsorption tests on soils. It would change the adsorption properties.